Page 149 - Physico-Chemical Niche Conditions for Bone Cells
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and 1 for a line segment. There was a higher percentage of cells with an eccentricity value larger than 0.9 and a lower percentage of cells with an eccentricity value smaller than 0.8 on SLBs with or without RGD than on PLL (p < 0.005). (e) Extent, a measure for the number of extensions, has a value between 0 and 1. With an increasing number of extensions, the extent value decreases. There was a lower percentage of cells with an extent value lower than 0.25 and a higher percentage of cells with an extent value between 0.25 and 0.5 on PLL then on SLBs with or without RGD (p < 0.05). (f) FormFactor, a measure for cell shape (4π ´ area/perimeter; Shah et al., 2019, equals 1 for a perfect circle). On all substrates ±80% of the cells had a FormFactor < 0.2 indicating that cells were elongated. There were no differences between substrates. n = 4 separate experiments, 20–140 cells/substrate/experiment (in total 80–560 cells/experiment) **p < 0.001, *p < 0.05. PLL, poly-L-lysine coated glass; SLBs, supported lipid bilayers.
Focal adhesion formation
Focal adhesion formation in MC3T3-E1 pre-osteoblasts on the different substrates was investigated by measuring the number and size of phosphorylated paxillin clusters, as a measure for the number and size of focal adhesions formed, and the intensity of integrin a5 staining as a measure for the number of integrins present in the cell (Figure 5). Cells on SLBs with or without RGD showed less and smaller clusters of phospho-paxillin than cells on PLL- coated glass (Figure 5b, c; p < 0.001). Cells cultured on SLBs with 0.5 μM RGD showed more phospho-paxillin clusters than cells cultured on SLBs with 0.2 μM RGD or 1.0 μM RGD (Figure 5c; p < 0.05). No differences in integrin a5 staining intensity between substrates were observed (Figure 5d). There were no differences in the levels of paxillin mRNA (both transcript variant a and β) between substrates (Figure 5e, f). ITGA5 mRNA content did not differ between substrates (Figure 5g).
Proliferation
To investigate whether RGD-functionalized SLBs influence proliferation of MC3T3-E1 pre- osteoblasts we investigated gene expression of Ki67 and Cyclin D1 (CCND1) (Figure 6). After 17 hr, the expression levels of both Ki67 and CCND1 in cells cultured on SLBs without RGD or on SLBs with 0.5 or 1.0 μM RGD were similar to those in cells cultured on PLL-coated glass (Figure 6a). There was a trend toward upregulation of Ki67 and CCND1 expression on SLBs with 0.2 μM RGD compared to SLBs without RGD (not significant). After 1 week, Ki67 and CCND1 expression did not differ between substrates, and were lower than after 17 hr of culture (Figure 6b).
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